The present invention is directed to formulations and methods for making drug-associated lipid complexes at high drug:lipid ratios (high drug:lipid complexes, or HDLCs). Such formulations are generally substantially equivalent or greater in efficacy to the same drug in their free form, yet have lower toxicity. Additionally, methods for the formation of such HDLCs are disclosed. More particularly, the invention is directed to the use of these high drug:lipid complexes with the toxic antifungal polyene antibiotics, specifically, amphotericin B and nystatin.
The high drug:lipid complexes (HDLCs) of the present invention can be made by techniques substantially the same as those for making liposomes. The invention includes the use of these HDLC structures in association with bioactive agents such as drugs, specifically the polyene antibiotics such as amphotericin B and nystatin.
As another aspect of the invention, a novel method for forming liposomes (or HDLCs) without the use of organic solvents is disclosed. Entrapment or association of a drug into the liposomes proceeds via an ethanol or an aqueous intermediate.
Liposomes are completely closed lipid bilayer membranes containing an entrapped aqueous volume. Liposomes may be unilamellar vesicles (possessing a single membrane bilayer) or multilamellar vesicles (onion-like structures characterized by multiple membrane bilayers, each separated from the next by an aqueous layer). The bilayer is composed of two lipid monolayers having a hydrophobic xe2x80x9ctailxe2x80x9d region and a hydrophilic xe2x80x9cheadxe2x80x9d region. The structure of the membrane bilayer is such that the hydrophobic (nonpolar) xe2x80x9ctailsxe2x80x9d of the lipid monolayers orient toward the center of the bilayer while the hydrophilic xe2x80x9cheadsxe2x80x9d orient towards the aqueous phase.
The original liposome preparation of Bangham et al. (J. Mol. Biol., 1965, 13:238-252) involves suspending phospholipids in an organic solvent which is then evaporated to dryness leaving a phospholipid film on the reaction vessel. Next, an appropriate amount of aqueous phase is added, the mixture is allowed to xe2x80x9cswell,xe2x80x9d and the resulting liposomes which consist of multilamellar vesicles (MLVs) are dispersed by mechanical means. This technique provides the basis for the development of the small sonicated unilamellar vesicles described by Papahadjopoulos et al. (Biochem. Biophys. Acta., 1967, 135:624-638), and large unilamellar vesicles.
Techniques for producing large unilamellar vesicles (LUVs), such as, reverse phase evaporation, infusion procedures, and detergent dilution, can be used to produce liposomes. A review of these and other methods for producing liposomes may be found in in the text Liposomes, Marc Ostro, ed., Marcel Dekker, Inc., New York, 1983, Chapter 1, the pertinent portions of which are incorporated herein by reference. See also Szoka, Jr. et al., (1980, Ann. Rev. Biophys. Bioeng., 9:467), the pertinent portions of which are also incorporated herein by reference. A particularly preferred method for forming LUVs is described in Cullis et al., PCT Publication No. 87/00238, Jan. 16, 1986, entitled xe2x80x9cExtrusion Technique for Producing Unilamellar Vesiclesxe2x80x9d incorporated herein by reference. Vesicles made by this technique, called LUVETS, are extruded under pressure through a membrane filter. Vesicles may also be made by an extrusion technique through a 200 nm filter; such vesicles are known as VET200s. These vesicles may be exposed to at least one freeze and thaw cycle prior to the extrusion technique; this procedure is described in Mayer et al., 1985, Biochem. et. Biophys. Acta., 817:193-196, entitled xe2x80x9cSolute Distributions and Trapping Efficiencies Observed in Freeze-Thawed Multilamellar Vesicles,xe2x80x9d relevant portions of which are incorporated herein by reference.
In the practice of this invention, a class of liposomes and method for their formation, characterized as having substantially equal lamellar solute distribution is preferred. This preferred class of liposomes is denominated as stable plurilamellar vesicles (SPLV) as defined in U.S. Pat. No. 4,522,803 to Lenk et al. and includes monophasic vesicles as described in U.S. Pat. No. 4,588,578 to Fountain et al. and frozen and thawed multilamellar vesicles (FATMLV) as described above.
A variety of sterols and their water soluble derivatives have been used to form liposomes; see specifically Janoff et al., U.S. Pat. No. 4,721,612, issued Jan. 26, 1988, entitled xe2x80x9cSteroidal Liposomesxe2x80x9d. Mayhew et al., PCT Publication No. WO 85/00968, published Mar. 14, 1985, describe a method for reducing the toxicity of drugs by encapsulating them in liposomes comprising alpha-tocopherol and certain derivatives thereof. Also, a variety of tocopherols and their water soluble derivatives have been used to form liposomes; see Janoff et al., PCT Publication No. 87/02219, published Apr. 23, 1987, entitled xe2x80x9cAlpha Tocopherol-Based Vesiclesxe2x80x9d and incorporated herein by reference.
In a liposome-drug delivery system, the bioactive agent such as a drug is entrapped in the liposome and then administered to the patient to be treated. For example, See Rahman et al., U.S. Pat. No. 3,993,754; Sears, U.S. Pat. No. 4,145,410; Papahadjopoulos et al., U.S. Pat. No. 4,235,871; Schneider, U.S. Pat. No. 4,224,179; Lenk et al., U.S. Pat. No. 4,522,803; and Fountain et al., U.S. Pat. No. 4,588,578. Alternatively, if the drug is lipophilic, it may associate with the lipid bilayer. In the present invention, the terms xe2x80x9centrapxe2x80x9d or xe2x80x9cencapsulatexe2x80x9d shall be taken to include both the drug in the aqueous volume of the liposome as well as drug associated with the lipid bilayer.
Many drugs that are useful for treating disease show toxicities in the patient; such toxicities may be cardiotoxicity, as with the antitumor drug doxorubicin, or nephrotoxicity, as with the aminoglycoside or polyene antibiotics such as amphotericin B. Amphotericin B is an extremely toxic antifungal polyene antibiotic, but the single most reliability in the treatment of life-threatening fungal infections (Taylor et al., Am. Rev. Respir. Dis., 1982, 125:610-611). Because amphotericin B is a hydrophobic drug, it is insoluble in aqueous solution and is commercially available as a colloidal dispersion in desoxycholate, a detergent used to suspend it which in itself is toxic. Amphotericin B methyl ester and amphotericin B have also been shown to be active against the HTLV-III/LAV virus, a lipid-enveloped retrovirus, shown in the etiology of acquired immuno-deficiency syndrome (AIDS) (Schaffner et al., Biochem, Pharmacol., 1986, 35:4110-4113). In this study, amphotericin B methyl ester ascorbic acid salt (water soluble) and amphotericin B were added to separate cultures of HTLV-III/LAV infected cells and the cells assayed for replication of the virus. Results showed that amphotericin B methyl ester and amphotericin B protected target cells against the cytopathic effects of the virus, similar to that demonstrated for the herpes virus (Stevens et al., Arch. Virol., 1975, 48:391).
Reports of the use of liposome-encapsulated amphotericin B have appeared in the literature. Juliano et al. (Annals N. Y. Acad. Sci., 1985, 446:390-402) discuss the treatment of systemic fungal infections with liposomal amphotericin B. Such liposomes comprise phospholipid, for example dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) in a 7:3 mole ratio, and cholesterol. Acute toxicity studies (LD50s) and in vitro assays comparing free and liposome-entrapped amphotericin B showed lower toxicities using the liposomal preparations with substantially unchanged antifungal potency. Lopez-Berestein et al. (J. Infect. Dis., 1986, 151:704-710) administered liposome-encapsulated amphotericin B to patients with systemic fungal infections. The liposomes comprised a 7:3 mole ratio of DMPC:DMPG, and the drug was encapsulated at a greater than 90% efficiency. As a result of the liposomal-drug treatment at 5 mol % amphotericin B, 66% of the patients treated responded favorably, with either partial or complete remission of the fungal infection. Lopez-Berestein et al. (J. Infect. Dis., 1983, 147:939-945), Ahrens et al., (S. Jour. Med. Vet. Mycol., 1984, 22:161-166), Panosian et al. (Antimicrob. Agents Chemo., 1984, 25:655-656), and Tremblay et al. (Antimicrob. Agents Chemo., 1984, 26:170-173) also tested the comparative efficacy of free versus liposomal amphotericin B in the treatment and prophylaxis of systemic candidiasis and leishmaniasis (Panosian et al., supra.) in mice. They found an increased therapeutic index with the liposome-encapsulated amphotericin B in the treatment of candidiasis. In all cases, it was found that much higher dosages of amphotericin B may be tolerated when this drug is encapsulated in liposomes. The amphotericin B-liposome formulations had little to no effect in the treatment of leishmaniasis.
Proliposomes (lipid and drug coated onto a soluble carrier to form a granular material) comprising DMPC:DMPG, ergosterol, and amphotericin B have also been made (Payne et al., J. Pharm. Sci., 1986, 75:330-333).
In other studies, intravenous treatment of cryptococcosis in mice with liposomal amphotericin B was compared to similar treatment with amphotericin B-desoxycholate (Graybill et al., J. Infect. Dis., 1982, 145:748-752). Mice treated with liposomal-amphotericin B showed higher survival times, lower tissue counts of cryptococci, and reduced acute toxicity. Multilameller liposomes used in this study contained ergosterol. Taylor et al. (Am. Rev. Resp. Dis., 1982, 125:610-611) treated histoplasmosis in mice with liposomal-amphotericin B wherein the liposomes contained ergosterol and phospholipids. The liposomal preparations were less toxic, more effective in treating histoplasmosis, and had altered serum and tissue distributions, with lower serum levels and higher liver and spleen concentrations than that of the free amphotericin B preparations.
In the above-mentioned studies, lipid-containing liposomes were used to ameliorate the toxicity of the entrapped drug, with the trend towards increasing the lipid content in the formulations in order to buffer drug toxicity. Applicants have surprisingly found that in fact a low lipid constituent decreases the toxicity most efficiently. In the formation of the HDLCs of the invention by an MLV method, a mixed population of HDLCs with MLVs can result; these preparations are those employing from about 6 to about 25 mole percent of drug (amphotericin B), with the proportion of HDLCs increasing as the mole percent drug increases. Preparations employing 25 mole percent to about 50 mole percent of drug are substantially HDLCs, free of liposomes. Alternatively, preparations containing 5 mole percent hydrophobic drug and less are substantially liposomal with some HDLCs. The separation of HDLCs from heterogenous populations if necessary, can be performed using any separation technique known in the art, for example, density gradient centrifugation.
The processes used to form these HDLCs can be substantially the same as those used to form liposomes, but in the present invention using high drug:lipid ratios, more HDLCs than liposomes are formed with unexpectedly large reduction in toxicity, compared to the liposomal formulations.
The present invention discloses HDLC (high drug:lipid ratio complexes) systems which comprise lipids and bioactive agents including drugs. Such HDLCs may comprise phospholipids such as DMPC and DMPG, preferably in a 7:3 mole ratio or saturated phospholipids or fatty acid phospholipids. The bioactive agent is preferably a drug, such as an antifungal drug such as nystatin or amphotericin B. The mole percent of the drug present is preferably from about 6 to about 50 mole percent, preferably 30 to 50 mole percent. Pharmaceutical compositions of the HDLCs, preferably comprising a drug such as amphotericin B, are made comprising pharmaceutical acceptable carriers or diluents, and these compositions may be administered parenterally. Such compositions are used to treat infectious diseases such as fungal infections, by administering them to mammals such as humans. The HDLC-containing compositions of the present invention include those compositions substantially free of liposomes and compositions substantially free of liposomes entrapping the drug. The term xe2x80x9csubstantially freexe2x80x9d shall be taken to mean generally no more than about 10 percent by weight of liposomes, preferably no more than about 5%, and more preferably no more than about 3%.
Various methods for preparing the HDLCs of the invention are disclosed; for example, techniques that first solubilize the drug, specifically amphotericin B in a solvent such as DMSO or methanol The lipid (preferably DMPC:DMPG in a 7:3 mole ratio) is solubilized in a solvent such as methylene chloride, and the lipid and drug solutions mixed. The solvents may be evaporated under reduced pressure, resulting in a thin lipid-drug film. The film may be hydrated in an aqueous solution such as saline, PBS, or glycine buffer, forming HDLCs. Alternatively, the aqueous solution may be added to the solvent-containing drug and lipid phase prior to evaporation of the solvent. As another alternative, the resulting dry lipid-drug film may be resuspended in a solvent, such as methylene chloride and again evaporated under reduced pressure prior to hydrating the film. A dehydration procedure may also be used; in this process a dry lipid-drug film is dehydrated to form a flake which is hydrated with aqueous solution.
In an alternative method for forming the HDLCs of the invention, lipid particles (or liposomes) containing bioactive agent (drug, for example polyene antifungals such as amphotericin B) made by the MLV process containing about 6 percent to 50 mole percent amphotericin B are formed and then the particles (or liposomes) are subjected to a heating cycle, at about 25xc2x0 C. to about 60xc2x0 C., most preferably about 60xc2x0 C. Such a cycle forms a more highly ordered and less toxic amphotericin B/lipid complex.
In another aspect of the invention, an absorbance spectrum technique is used to determine the toxicity of a drug (e.g. a polyene antifungal such as amphotericin B)-lipid complex. The absorbance spectrum of a drug is specific for that drug; the signature of the drug may be a peak or series of peaks in the ultraviolet or the visible range. The signature peak for amphotericin B, (appearing in FIG. 12, dissolved in deoxycholate), is between 300 and 500 nm, and has characteristic peaks, the most representative of these peaks being the one arising at 413 nm. The attenuation of this peak by complexing the drug with lipid can be used quantitatively as a measure of toxicity of the HDLC. In other words, the degree of toxicity may be determined by the intensity of the absorbance peak height.
A liposome-loading process is also disclosed wherein the drug, specifically the polyene antibiotic amphotericin B is dispersed by sonication in a solvent such as ethanol to which has been added an acid such as hydrochloric acid. A lipid film, specifically comprising DMPC:DMPG in an about 7:3 mole ratio, is hydrated with an aqueous solution, specifically aqueous buffer such as PBS, and an aliquot of the acidified ethanol solution containing the drug is loaded into the liposomes by adding it to the liposome preparation. The ethanol in the resulting suspension is removed and the solution is resuspended with an aqueous solution. Depending on the mole ratio of drug co-mixed with the lipid, the process favors formation of HDLCs rather than liposomes; e.g. at mole percent of drug of about 16 and above, more HDLCs are formed than liposomes. Alternatively, at 0-15 mole percent drug, the process favors formation of liposomes. Liposomes or HDLCs made by this acidified ethanol loading process may be prepared for use as pharmaceutical compositions by the addition of pharmaceutically acceptable carriers or diluent, and may be used in the treatment of fungal infections by administering them to a mammal such as a human.